Glycosyl hydrolase xylanases, compositions and methods of use for efficient hydrolysis and processing of xylan

ABSTRACT

The invention provides a unique subset of GH30 subfamily 8 xylanases (GH30-8) with endo-β-1,4-xylanase activity, compositions comprising an effective amount of the GH30-8 xylanases, methods of synthesis and methods of use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/023,116, filed Jul. 10, 2014, which is incorporated by reference inits entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention is owned by and was made with government support from theUSDA Forest Service. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Xylanases (endo-1,4-β-xylanase, EC 3.2.1.8) hydrolyze internalβ-1,4-xylosidic linkages in xylan to produce smaller molecular weightxylose and xylo-oligomers. Xylans are polysaccharides formed from1,4-β-glycoside-linked D-xylopyranoses. Xylanases are very useful inmultiple commercial applications, including for dough preparation orbread product preparation fruit and vegetable processing, breaking downagricultural waste, manufacturing animal feed as well as inlignocellulose pretreatment and pulp and paper production. Cellulose andhemicellulose materials which can be converted into fermentable sugarsare considered a very useful and under-utilized source of renewablebiomass materials. Individual β-1,4-glucose chains once synthesized,self-associate through hydrogen bonding to form semi-crystallinecellulose microfibrils (cellulose). The cellulose microfibrils areembedded in a polysaccharide matrix formed of hemicelluloses such asxylan, galactoglucomannan and xyloglucan all of which may be associatedwith other lower abundance biomass polysaccharides such as arabinans,mannans; pectins including galacturonans and galactans and various otherβ-1,3 and β-1,4 glucans all of which are dependent upon the plant sourceand the cellulosic tissue in question. The hemicellulose matrix is alsotypically surrounded and cross linked with polyphenolic lignins. Fromthe tight interactions that exist between cellulose, hemicellulose andlignin, it is very difficult and expensive to break down thisrecalcitrant matrix of biomass to yield desired mixtures ofoligosaccharides or fermentable simple sugars.

The primary hemicellulose from hardwood and crop residues is aglucuronoxylan (GX) consisting of a chain of β-1,4-linked xyloseresidues randomly substituted with α-1,2-linked glucuronic acid (GA)residues. Frequency of substitution has been shown to be as high as 1 GAfor every 6 xyloses. In hardwoods, unaltered GX is additionallyacetylated to a high degree on the O-2 or O-3 (or O-2 and O-3) hydroxylpositions. Commercial extraction of these polysaccharides typically isdone under alkaline conditions which deacetylates these polysaccharides.This is the form of GX commonly used for laboratory studies. Other loweryielding extraction procedures must be implemented to obtain aglucuronoacetylxylan polysaccharide. Xylan is the second most abundanthemicellulose in softwood species next to galactoglucomannan, accountingfor just less than half of the total hemicellulose. This source of xylanis in the form of glucuronoarabinoxylan with periodic GA substitutionsand α-1,2-linked arabinofuranose substitution on the O-2 or O-3 (or O-2and O-3) hydroxyl positions of xylose. Xylans from other sources such asgrains (wheat, WAX) typically consist of an arabinoxylan, havingpatterns of arabinofuranose substitution similar to softwoods, butlacking the GA substitution.

The glycosyl hydrolase (GH) family 30 (GH30) enzymes (Cantarel, et al.,2009) have recently been redefined (St. John, et al., 2010). The newfamily composition consists of eight subfamilies that can be assignedinto two structurally and phylogenetically distinguishable groups.Biochemical and structural studies have shown the enzymes in subfamily 8to have unique characteristics in the degradation of the hemicellulosicpolymer glucuronoxylan (St. John, et al., 2006; Vrsanska, et al., 2007;Hurlbert & Preston, 2001).

Accordingly, a need exists for novel glycosyl hydrolase enzymes,compositions and methods of use which can more efficiently convert plantor other cellulosic or hemicellulosic materials into fermentable sugars.

SUMMARY OF THE INVENTION

The present invention provides a functionally unique subset of GH30subfamily 8 xylanases (GH30-8) with GA-independent endo-β-1,4-xylanaseactivity, compositions comprising an effective amount of the GH30-8xylanases of the present invention, methods of synthesis and methods ofuse thereof.

In a first aspect, the invention encompasses an isolated GA-independentGH30-8 enzyme or variant thereof exhibiting xylanase activity. Theenzyme or variant includes the amino acid sequence (W or Y)(W or F)W(Ior W or F)(not R)(not R) within the β8-α8 loop of the enzyme or variant.

In an alternate aspect, the invention comprises an isolatedGA-independent GH30-8 enzyme or variant thereof exhibiting xylanaseactivity comprising at least one of SEQ ID NOs: 1-4 in the β7-α7 andβ8-α8 loops, wherein the amino acid sequence (W or Y)(W or F)W(I or W orF)(not R)(not R) is within the β8-α8 loop of the enzyme or variant.

In an alternate aspect, the invention comprises an isolatedGA-independent GH30-8 enzyme exhibiting xylanase activity or variantcomprising an amino acid sequence that at least 30% similar to an aminoacid sequence selected from the group consisting of G7M3Z8, MINOD3,Q97TI2, F7ZYN8, FOKEL6, COIQA1, COIQA2, B3TJG3, E4T7O5, H1YFT8, andF1TBY8.

In some embodiments, GA-independent GH30-8 enzyme or variant of theinvention includes an amino acid sequence that at least 30% similar toSEQ ID NO:1 residues 33-420 (Q97TI2), SEQ ID NO:2 residues 32-421(F1TBY8), SEQ ID NO:3 beginning at residue 45 (E4T705), SEQ ID NO:4beginning at residue 33 (H1YFT8), SEQ ID NO:32 (C0IQA1), SEQ ID NO:33(B3TJG3), SEQ ID NO:34 (G7M3Z8), SEQ ID NO:35 (M1N0D3), SEQ ID NO:36(F7ZYN8), or SEQ ID NO:37 (F0KEL6). In some such embodiments, theGA-independent GH30-8 enzyme or variant includes an amino acid sequencethat is at least 80% similar to the selected amino acid sequence. Insome such embodiments, the GA-independent GH30-8 enzyme or variantincludes an amino acid sequence that is at least 95% similar to theselected amino acid sequence.

In some embodiments, the GA-independent GH30-8 enzyme or variantincludes the amino acid sequence of SEQ ID NO:1 residues 33-420(Q97TI2), SEQ ID NO:2 residues 32-421 (F1TBY8), SEQ ID NO:3 beginning atresidue 45 (E4T705), SEQ ID NO:4 beginning at residue 33 (H1YFT8), SEQID NO:32 (C0IQA1), SEQ ID NO:33 (B3TJG3), SEQ ID NO:34 (G7M3Z8), SEQ IDNO:35 (M1N0D3), SEQ ID NO:36 (F7ZYN8), SEQ ID NO:37 (F0KEL6), or theamino acid sequence of C0IQA2.

In some embodiments, the GA-independent GH30-8 enzyme is XynQ97.

In some embodiments, GA-independent GH30-8 enzyme is XynC71 (akaCpXyn30A).

In a second aspect, the invention encompasses a GH30-8 enzymecomposition. The composition includes a first polypeptide havingxylanase activity that includes the amino acid sequence (W or Y)(W orF)W(I or W or F)(not R)(not R), and a second polypeptide different fromthe first having xylanase activity that also includes the amino acidsequence (W or Y)(W or F)W(I or W or F)(not R)(not R). The GH30-8 enzymecomposition is capable of hydrolyzing a lignocellulosic biomassmaterial.

In some embodiments, the composition further includes at least oneadditional protein having enzymatic activity.

In some embodiments, the first polypeptide, the second polypeptide, orboth include an amino acid sequence that at least 30% similar to SEQ IDNO:1 residues 33-420 (Q97TI2), SEQ ID NO:2 residues 32-421 (F1TBY8), SEQID NO:3 beginning at residue 45 (E4T705), SEQ ID NO:4 beginning atresidue 33 (H1YFT8), SEQ ID NO:32 (C0IQA1), SEQ ID NO:33 (B3TJG3), SEQID NO:34 (G7M3Z8), SEQ ID NO:35 (M1N0D3), SEQ ID NO:36 (F7ZYN8), or SEQID NO:37 (F0KEL6). In some such embodiments, the first polypeptide, thesecond polypeptide, or both include an amino acid sequence that is atleast 80% similar to the selected amino acid sequence. In some suchembodiments, the first polypeptide, the second polypeptide, or bothinclude an amino acid sequence that is at least 95% similar to theselected amino acid sequence.

In some embodiments, the first polypeptide, the second polypeptide, orboth include an amino acid sequence selected from SEQ ID NO:1 residues33-420 (Q97TI2), SEQ ID NO:2 residues 32-421 (F1TBY8), SEQ ID NO:3beginning at residue 45 (E4T705), SEQ ID NO:4 beginning at residue 33(H1YFT8), SEQ ID NO:32 (C0IQA1), SEQ ID NO:33 (B3TJG3), SEQ ID NO:34(G7M3Z8), SEQ ID NO:35 (M1N0D3), SEQ ID NO:36 (F7ZYN8), SEQ ID NO:37(F0KEL6), or the amino acid sequence of COIQA2.

In some embodiments, the amount of polypeptides having xylanase activityrelative to the total amount of proteins in the enzyme composition isabout 10 wt. % to about 20 wt. %.

In a third aspect, the disclosure encompasses a method of hydrolyzing ordigesting a lignocellulosic biomass material comprising hemicelluloses,cellulose, or both. The method includes the steps of contacting theGH30-8 enzyme composition described above with the lignocellulosicbiomass mixture.

In some embodiments, the lignocellulosic biomass mixture comprises anagricultural crop, a byproduct of a food/feed production, alignocellulosic waste product, a plant residue, or waste paper.

In some embodiments, the biomass material in the lignocellulosic biomassmixture is subjected to pretreatment, wherein the pretreatment is anacidic pretreatment or a basic pretreatment. In some such embodiments,the pretreament includes a thermal, aqueous or thermomechanical pulping.

In some embodiments, the GH30-8 enzyme composition is used in an amountand under conditions and for a duration sufficient to convert at least60% to 90% of the xylan in the biomass material intoxylooligosaccharides.

Other objects, features and advantages of the present invention willbecome apparent after review of the specification, claims and drawings.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, and patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood and features, aspectsand advantages other than those set forth above will become apparentwhen consideration is given to the following detailed descriptionthereof.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. Exemplified glucuronoxylan processing by canonical GA-dependentGH30-8 xylanases.

FIG. 2. Molecular contacts between aldotriuronate and the activesite/substrate binding cleft of the canonical GH30-8glucuronoxylanhydrolase, XynC from Bacillus subtilis 168.

FIG. 3. A close-up of the glucoronate appendage with the typicallyconserved β7-α7 and β8-α8 loop regions of XynC of Bacillus subtilisshowing the contacts which allow specificity for this appendage in thecanonical GH30-8 xylanases.

FIG. 4. An amino acid alignment of diverse GA-dependent GH30-8 xylanaseamino acid sequences with similarity to XynC from Bacillus subtilis andXynA from Erwinia chrysanthemi and showing the canonical conservedregions between these enzymes which includes the β7-α7 and β8-α8 loopregions. The sequence labeled Q45070 is SEQ ID NO:5; the sequencelabeled V3T509 is SEQ ID NO:6; the sequence labeled S9R6D6 is SEQ IDNO:7; the sequence labeled Q4URA6 is SEQ ID NO:8; the sequence labeledB3PEH7 is SEQ ID NO:9; the sequence labeled U4MR52 is SEQ ID NO:10; thesequence labeled M1MG04 is SEQ ID NO:11; the sequence labeled R7H6D7 isSEQ ID NO:12; the sequence labeled W4C6X9 is SEQ ID NO:13; the sequencelabeled D7C0B1 is SEQ ID NO:14; the sequence labeled M1MLA3 is SEQ IDNO:15; the sequence labeled D3EH02 is SEQ ID NO:16; the sequence labeledU4QXC0 is SEQ ID NO:17; the sequence labeled IOL743 is SEQ ID NO:18; thesequence labeled K2P3H7 is SEQ ID NO:19; the sequence labeled E3E322 isSEQ ID NO:20; the sequence labeled M5RGV9 is SEQ ID NO:21; the sequencelabeled A8FDV2 is SEQ ID NO:22; the sequence labeled B4ADC5 is SEQ IDNO:23; the sequence labeled R9TYN3 is SEQ ID NO:24; the sequence labeledM1XAU4 is SEQ ID NO:25; the sequence labeled C9RS22 is SEQ ID NO:26; thesequence labeled F4FBT9 is SEQ ID NO:27; the sequence labeled E8SBF5 isSEQ ID NO:28; the sequence labeled R6LF43 is SEQ ID NO:29; the sequencelabeled H1YFT7 is SEQ ID NO:30; the sequence labeled Q46961 is SEQ IDNO:31.

FIG. 5. An amino acid sequence alignment of the GA-independent GH30-8subset of enzymes being disclosed including the canonical GA-dependentenzymes XynC from Bacillus subtilis and XynA from Erwinia chrysanthemifor comparison revealing how these enzymes show high levels of sequenceidentity not different than that shown in FIG. 4, yet the β7-α7 andβ8-α8 loop regions are conserved. The sequence labeled Q45070 in FIG. 5is residues 2-389 of SEQ ID NO:5, which ends with amino acids ofasparagine (Asn or N) and arginine (Arg or R); the sequence labeledC0IQA1 is SEQ ID NO:32; the sequence labeled B3TJG3 is SEQ ID NO:33; thesequence labeled E4T705 is SEQ ID NO:3, beginning at residue 45; thesequence labeled H1YFT8 is SEQ ID NO:4, beginning at residue 33; thesequence labeled G7M3Z8 is SEQ ID NO:34; the sequence labeled M1N0D3 isSEQ ID NO:35; the sequence labeled Q97TI2 is SEQ ID NO:1, residues 33 to420; the sequence labeled F7ZYN8 is SEQ ID NO:36; the sequence labeledF0KEL6 is SEQ ID NO:37; the sequence labeled F1TBY8 is SEQ ID NO:2,residues 34 to 421; the sequence labeled Q46961 is SEQ ID NO:31, whichends with a lysine (Lys or K).

FIG. 6. Limit hydrolysis product analysis by thin layer chromatography,comparing the hydrolysis products of the GA-independent CpXyn30A (C7I)and CaXyn30A (Q97) enzymes to the canonical GA-dependent GH30-8 xylanaseXynC from Bacillus subtilis and a GH10 family xylanase.

FIG. 7A. Small scale sequence alignment highlighting the β7-α7 and β8-α8region of the GA-dependent GH30-8 enzymes with the GA-independentCpXyn30A (C7I, Cp_F1TBY8) included for comparison. The sequence labeledBs_Q45070 is SEQ ID NO:5, residues 223-295; the sequence labeledCs_M1MLA3 is SEQ ID NO:15, residues 223-295; the sequence labeledCp_F1TBY8 is SEQ ID NO:2, residues 258 to 324; The sequence labeledRa_E6UFE8 is SEQ ID NO:38; the sequence labeled Cj_B3PEH7 is SEQ IDNO:9, residues 220 to 292; the sequence labeled Xp_FOBZ40 is SEQ IDNO:39; the sequence labeled Ec_Q46961 is SEQ ID NO:31, residues 219 to288.

FIG. 7B. The native DNA coding sequence of CpXyn30A encodes a protein of628 amino acids including a N-terminus secretion signal sequence, aC-terminal family 6 carbohydrate binding module and two C-terminaldockerin domains for interaction of the secreted, mature form ofCpXyn30A with a cellulosome complex.

FIG. 7C. In support of the qualitative sequence alignment in FIG. 7A, aphylogenetic tree showing that CpXyn30A (Cp_F1TBY8) with its uniquesequence in the β7-α7 and β8-α8 loop regions still groups confidentlywith the canonical GH30-8 enzymes.

FIG. 8A. The crystal structure of CpXyn30A superposed on XynC ofBacillus subtilis establishing a structural relationship.

FIG. 8B. CpXyn30A superposed with XynC from Bacillus subtilis to comparestructural differences in the α2 and α3 helix region.

FIG. 8C. CpXyn30A superposed with XynC from Bacillus subtilis to comparethe β3-α3 loop region which indicate that this notable structuralportion is more similar to GH30-8 enzymes which derive fromGram-positive bacteria.

FIG. 9A. Ligand bound structural model of XynC from Bacillus subtilissuperposed with the structure model of CpXyn30A showing how thesubstrate binding specificity is altered by the changes to the β7-α7 andβ8-α8 loops.

FIG. 9B. Superposition of CpXyn30A with XynC from Bacillus subtilisshowing the amino acid side chains which are different resulting in aloss of specificity.

FIG. 10A. Thin layer chromatography analysis of hydrolysis products ofglucuronoxylan (SGX) and arabinoxylan (WAX) by CpXyn30A compared withXynC from Bacillus subtilis.

FIG. 10B. Thin layer chromatography analysis of hydrolysis products ofxylooligosaccharides by CpXyn30A compared to XynC from Bacillussubtilis.

FIG. 11. HPLC analysis of CpXyn30A hydrolysis of xylohexaose showingtransglycosylation activity and a flow chart for how this occurs.

FIG. 12. β7-α7 loop regions of the disclosed GH30-8 subset of enzymesbeing compared to XynC from Bacillus subtilis (top, Q45070_Bsubtili) andXynA from Erwinia chrysanthemi (bottom, Q46961_Dchrysan). The sequencelabeled Q45070 is SEQ ID NO:5, residues 222 to 241; the sequence labeledC0IQA1 is SEQ ID NO:32, residues 218 to 235; the sequence labeled B3TJG3is SEQ ID NO:33, residues 214 to 231; The sequence labeled E4T705 is SEQID NO:3, residues 255 to 275; the sequence labeled H1YFT8 is SEQ IDNO:4, residues 234 to 252; the sequence labeled G7M3Z8 is SEQ ID NO:34,residues 219 to 236; the sequence labeled M1N0D3 is SEQ ID NO:35,residues 219 to 236; the sequence labeled Q97TI2 is SEQ ID NO:1,residues 253 to 270; the sequence labeled F7ZYN8 is SEQ ID NO:36,residues 221 to 238; the sequence labeled F0KEL6 is SEQ ID NO:37,residues 221 to 238; the sequence labeled F1TBY8 is SEQ ID NO:2,residues 257 to 274; the sequence labeled Q46961 is SEQ ID NO:31,residues 218 to 235.

FIG. 13. B8-α8 loop regions of the disclosed GH30-8 subset of enzymesbeing compared to XynC from Bacillus subtilis (top, Q45070_Bsubtili) andXynA from Erwinia chrysanthemi (bottom, Q46961_Dchrysan). The sequencelabeled Q45070 is SEQ ID NO:5, residues 263 to 278; the sequence labeledC0IQA1 is SEQ ID NO:32, residues 253 to 270; the sequence labeled B3TJG3is SEQ ID NO:33, residues 249 to 268; The sequence labeled E4T705 is SEQID NO:3, residues 292 to 311; the sequence labeled H1YFT8 is SEQ IDNO:4, residues 269 to 287; the sequence labeled G7M3Z8 is SEQ ID NO:34,residues 254 to 270; the sequence labeled M1N0D3 is SEQ ID NO:35,residues 254 to 270; the sequence labeled Q97TI2 is SEQ ID NO:1,residues 288 to 304; the sequence labeled F7ZYN8 is SEQ ID NO:36,residues 256 to 272; the sequence labeled F0KEL6 is SEQ ID NO:37,residues 256 to 272; the sequence labeled F1TBY8 is SEQ ID NO:2,residues 292 to 307; the sequence labeled Q46961 is SEQ ID NO:31,residues 256 to 271.

FIG. 14. SEQ ID NO: 1. Amino acid sequence of UniProt accession Q97TI2having the named protein expression product Q97 (aka XynQ97, CaXynQ97,CaXyn30A, Q97_RCN and XynQ97_RCN).

FIG. 15. SEQ ID NO: 2. Amino acid sequence of UniProt accession F1TBY8having 100% identity to the obsolete UniProt accession C7IMC9 and havingthe named protein expression product C7I (aka XynC7I, CpXynC7I andCpXyn30A).

FIG. 16. SEQ ID NO: 3. Amino acid sequence of UniProt accession E4T705and having the named protein expression product PpXyn30A.

FIG. 17. SEQ ID NO: 4. Amino acid sequence of UniProt accession H1YFT8and having the named protein expression product MpXyn30A.

FIG. 18. The β2-α2 loop region of CpXyn30A showing a fine differencebetween the structure of this enzyme and the canonical GH30-8 subfamilyof enzymes.

FIG. 19. SDS-PAGE analysis of additional GA-independent GH30-8 xylanasesand the full CaXyn30A (Q97_RCN) xylanase containing the natively encodedC-terminal CBM13 module.

FIG. 20. TLC analysis of additional GA-independent GH30-8 xylanases andthe full CaXyn30A (Q97_RCN) xylanase containing the natively encodedC-terminal CBM13 module along with the originally studied Q97 catalyticdomain.

FIG. 21A. Schematic representing the xylosyl binding subsites of anendo-xylanase active site detailing the locations along the xylan chainwhere the enzyme may accommodate or require (as in the GA-dependentGH30-8 xylanases) a GA substitution. Example of GX within the activesite of a GH10 xylanase showing GA accommodating xylose subsites andtherefore a deduced limit product of aldotetrauronate.

FIG. 21B. Example of GX within the active site of a GH30-8 GAindependent xylanase showing GA accommodating xylose subsites andtherefore a deduced limit product of aldotriuronate.

DETAILED DESCRIPTION OF THE INVENTION I. In General

Before the present materials and methods are described, it is understoodthat this invention is not limited to the particular methodology,protocols, materials, and reagents described, as these may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”, and“having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications and patentsspecifically mentioned here are incorporated by reference for allpurposes including describing and disclosing the chemicals, instruments,statistical analysis and methodologies which are reported in thepublications which might be used in connection with the invention. Allreferences cited in this specification are to be taken as indicative ofthe level of skill in the art. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

II. The Invention

The present invention provides a functionally unique subset of GH30subfamily 8 xylanases (GH30-8) with endo-β-1,4-xylanase activity,compositions comprising an effective amount of the GH30-8 xylanases ofthe present invention, methods of synthesis and methods of use thereof.

GH30-8 Xylanases.

Glycoside hydrolase enzymes defined in family 30 (GH30) have at least 8subfamilies (GH30-1 through GH30-8). Enzymes classified into the GH30-8subfamily constitute a well characterized group of endoxylanases whichcleave the β-1,4-xylosidic linkage of xylan only upon recognition of theα-1,2-linked 4-O-methylglucuronic acid (glucuronic acid, GA) side chainappendage common to many xylan types and sources (St. John, et al.,2010; St. John, et al., 2006). Cleavage of the xylan chain occurs towardthe polymer reducing terminus relative to the target glucuronic acid,such that the GA appendage is positioned penultimate to the new reducingterminus.

Limit hydrolysis of glucuronoxylan by these “GA-dependent” xylanasesprimarily result in a distribution of aldouronates in which eachcontains a single GA appendage substituted on the xylose penultimate tothe reducing terminal of the resulting aldouronate (FIG. 1). The way inwhich these enzymes function to perform this specific type ofglucuronoxylan chain cleavage has been described through biochemicalstudies and more recently from a detailed understanding of the proteinstructure with associated xylan derived ligands (FIGS. 2 & 3) (St. Johnet al., 2011; Urbanikova et al., 2011).

Before the enzymes of the present invention were identified and pursued,it was strongly established and widely agreed upon that GH30-8 enzymeswere restricted to cleaving the β-1,4-linkage of xylan next to a GAsubstitution (FIGS. 1, 21). Inherent in that understanding is that themechanism which allowed for that to occur, if it were to not be what itis, then the enzyme may no longerenzyme will not function. It wastherefore well considered that in the absence of GA. Thus, thesecanonical GA-dependent GH30-8 xylanases, therefore requiredare known torequire GA for their function.

The presently disclosed, functionally distinct subset of GH30-8xylanases and compositions thereof have a relaxed (or expanded range of)substrate specificity which results in a gain of function, because tofunction, they do not require the O-2 linked GA. These “GA-independent”xylanases are thus able to hydrolyze diverse polymeric xylans, includingglucuronoxylans (GX) such as sweetgum wood xylan (SGX) and beech woodxylan (BX), arabinoxylans such as wheat arabinoxylan (WAX) and neutralxylooligosaccharide (e.g. X6) to smaller xylooligosaccharides andsubstituted xylooligosaccharides. Such classes of compositions cannot behydrolyzed by the typical GA-dependent GH30-8 xylanases, which requirethe O-2 linked glucuronic acid (FIG. 6).

Specifically, the GH30-8 GA-independent xylanases of the presentinvention comprise any protein which, through primary sequence analysis,contains a confidently classified GH30-8 catalytic module with familyconserved catalytic amino acids identified, as a part, or whole of amature amino acid sequence, but having an altered sequence in place ofthe functionally characterized, GH30-8 subfamily conserved β7-α7 andβ8-α8 loops of interest, as found in the XynQ97 (CaXyn30A), XynC7I(CpXyn30A), PpXyn30A and MpXyn30A xylanases and described furtherherein. In one embodiment, the specific amino acid sequence in the β7-α7and β8-α8 loops is as shown in SEQ ID NO: 1 (FIG. 5, Label:Q97TI2_Cacetobu & FIG. 14). The invention also includes thoseconfidently classified GH30-8 (as described above) xylanases comprisingat least 30% similarity, at least 40% similarity, at least 50%similarity, at least 60% similarity, at least 70% similarity, at least80% similarity, at least 90% similarity, at least 95% similarity, or atleast 98% similarity to the specific amino acid sequence in the β7-α7and β8-α8 loop regions of one or more of SEQ ID NO:1, SEQ ID NO:2 (C7I),SEQ ID NO:3 (PpXyn30A) or SEQ ID NO:4 (MpXyn30A).

While the GA-independent GH30-8 xylanases are different in the β7-α7 andβ8-α8 loop regions relative to the conserved sequence of these loops inthe GA-dependent GH30-8 xylanases, these sequences are also notablydiverse within this GH30-8 subset of xylanases (FIGS. 5, 12 and 13),highlighting a likelihood that they may all function uniquely. Thefunction of GA-dependent xylanases appears primarily attributable to theconserved β8-α8 loop sequence WW(YF)(IGL)(RK)R(SQYFC)Y(GS) (RR-motif)(as ascertained from the diverse alignment provided in FIG. 4). In theGH30-8 GA-independent xylanases the conserved RR-motif sequence in thisloop is replaced with the sequence (WY)(WF)W(IWF)(not R)(not R) (asascertained from the GA-independent GH30-8 alignment provided in FIG.5). Accordingly, the invention encompasses GA-independent GH30-8xylanases or variants thereof comprising the β8-α8 loop sequence(WY)(WF)W(IWF)(not R)(not R).

Indeed, the CpXyn30A (C7I) xylanase performed very similar to theCaXyn30A (Q97) with respect to the final hydrolysis products detected byTCL, but the measured rate of hydrolysis was notably low. It is notclear from our current level of analysis whether the CpXyn30A yielded aportion of larger oligosaccharides as observed in the TLC (FIG. 6) forboth beech wood xylan and wheat arabinoxylan because of this comparablylow activity, or because of the unique sequence differences between theβ7-α7 and β8-α8 loop regions of these two GA-independent GH30-8xylanases. Likewise, the PpXyn30A enzyme with yet a different sequencerepresenting its β7-α7 and β8-α8 loops produced very similar results toboth CpXyn30A and CaXyn30A (FIG. 20). However, the MpXyn30A enzyme,while clearly functioning as an endoxylanase yielding larger neutralxylooligosaccharides such as xylotriose and xylotetraose failed toresult in any significant hydrolysis (barely detectable) yet is stillshown to be GA-independent (FIG. 20).

The GA-independent GH30-8 xylanases of the present invention hydrolyzeGX to provide a series of small neutral xylooligosaccharides andaldouronates (FIGS. 6, 10A, 10B and 20), which is very different fromthe aldouronate ladder produced by the GA-dependent GH30-8 xylanases(FIGS. 6, 10A and 10B). Additionally the GX hydrolysis product profilefor the GA-independent GH30-8 xylanases of the present invention aremore similar, but unique to GX hydrolysis product profiles generated bythe common GH10 and GH11 endo-β-1,4-xylanases. None of theGA-independent GH30-8 xylanases result in xylose as a primary hydrolysisproduct as observed for some GH10 xylanases. Also, unlike the GH10 andGH11 xylanases which produce, as their primary aldouronate limit productof GX hydrolysis, the tetrameric product aldotetrauronate andaldopentauronate, respectively, the GA-independent GH30-8 enzymes arepotentially able to liberate aldotriuronate as the smallest limitproduct (FIGS. 21A-B).

Since hydrolysis of arabinoxylan by GA-dependent GH30-8 xylanases doesnot occur (FIG. 6), the hydrolysis product profile obtained with theGA-independent GH30-8 xylanases on this substrate is very unique.Further, these enzymes appear to be very efficient at the liberation ofdifficult to reduce (hydrolyze) arabinoxylan substrates, relative toGH10 xylanases (FIG. 6). For this substrate, the tested GA-independentGH30-8 xylanases produce unique hydrolysis product profiles.

Rationalization and our results support the likelihood that theGA-independent GH30-8 xylanases are better at liberating smallsubstituted xylooligosaccharides and substituted xylooligosaccharidesfrom highly substituted regions of xylans (FIGS. 21A-B). The currentstate-of-the-art is that GH10 xylanases produce the smallest hydrolysisproducts. The primary limit product of these xylanases isaldotetrauronate. This is because, as detailed in FIG. 21A the enzymecan accommodate the O-2 substituted GA moiety only in the +1 and −3subsites within the substrate binding cleft. In comparison to these GH10xylanases and, as detailed in FIG. 21B, for GA-dependent GH30-8xylanases the −2 subsite is responsible for the coordination of a GAsubstituted xylose. Without this, hydrolysis is known not to occur. Inthese enzymes, although the GA is specifically “bound” in this position,the general observation is that the GA moiety is extended upward out ofthe catalytic cleft and therefore not into the enzyme where stericinteraction might prevent hydrolysis. Notably, based on limit productstudies of GX by these GA-dependent GH30-8 xylanases, the smallestaldouronate that might be produced (depending upon the nature of thespecific GX being analyzed) is aldotriuronate. This would indicate thatin these xylanases, a GA substitution can also be accommodated on thexylose in the +1 subsite (see FIGS. 21A-B). Based on xylan chain bondingand the reported position within xylan binding enzymes, it appears verylikely that the xylose in this subsite would present the O-2 hydroxylupward out of the substrate binding cleft. A GA substituted in thisposition would likely therefore be accommodated.

Extending this rationale to the GA-independent GH30-8 xylanases of thepresent invention, it would than seem possible that, barring changes tothe +1 subsite region, both the +1 and −2 subsites may accommodatesubstituted GA moieties. This is verified with the CaXyn30A (Q97)xylanase (FIG. 6), as the sugar aldotriuronate is clearly visiblefollowing a limit digestion. The extended range of function of theseenzymes is also likely to benefit hydrolysis of the more highlysubstituted (with arbinofuranose) wheat arabinoxylan substrate. This isconfirmed by results in the hydrolysis of this substrate (FIG. 6), wherecompared to the GH10 xylanase CaXyn30A, it effectively converted theentire starting amount of xylan to xylooligosaccharides and substitutedxylooligosaccharides.

In one embodiment, the GA-independent, GH30-8 xylanases of the presentinvention represent eight (upon last count) nonredundent sequences outof several hundred in the UniProt protein database which through primarysequence analysis are confidently classified as GH30-8 xylanases.However, in each of these the sequence of the β7-α7 and β8-α8 loopregions is completely different than the canonical sequence found in theGA-dependent GH30-8 xylanases and also unique within the disclosedsubset as described above. These enzymes are shown to have a loss of GXsubstrate specificity and expanded function, providing unique xylanhydrolysis product profiles relative to the GA-dependent GH30-8xylanases and xylanases from other xylanase enzyme families and also areproposed to be more efficient in the hydrolysis of highly substitutedpolymeric xylan for reasons presented throughout.

In one embodiment, by “GA-independent GH30-8 xylanases” we mean theisolated enzymes having xylanase activity comprising amino acidsequences from Clostridium (UniProt accession numbers G7M3Z8, MINOD3,Q97TI2 {Q97}, F7ZYN8 and FOKEL6); the southern root knot nematodeMeloidogyne incognita (UniProt accession numbers COIQA1 and COIQA2); theplant pathogenic nematode Radopholus simitis (UniProt accession numberB3TJG3); the bacterium Paludibacter propionicigenes (UniProt accessionnumber E4T705); the bacterium Mucilaginibacter paludis (UniProtaccession number H1YFT8); and from Clostridium papyrosolvens (UniProtaccession number F1TBY8 {C7I}).

In one embodiment, the GH30-8 enzymes are CpXyn30A (also referred tothroughout as C7I, XynC7I and CpXynC7I) and CpXynQ97 (also referred tothroughout as Q97 and XynQ97 and CaXynQ97).

By “isolated enzyme” we mean polypeptides isolated from other cellularproteins, purified and recombinant polypeptides, cellular material,viral material, chemical precursors or other chemicals.

TABLE 1 Comparison of amino acid identity levels of the GH30-8 GA-independent xylanases of the present invention to the characterizedGH30-8 GA-dependent xylanases BcXynC and EcXynA. Level of Identity tothe Canonical GH30-8 Xylanases (%)¹ XynC XynA XynQ97 (UniProt: (UniProt:(UniProt: 45070) Q46961) Q97TI2)² Q45070_Bsubtilis (BsXynC) 100 40.440.9 C0IQA1_Mincognita 34.3 34.6 38.9 B3TJG3_Rsimilis 38.8 45.0 47.2E4T705_Ppropionicigenes 37.5 41.6 42.1 (PpXyn30A)² H1YFT8_Mpaludis(MpXyn30A)² 38.2 35.4 39.7 G7M3Z8_Clostridium sp. 43.6 43.5 65.6M1N0D3_Csaccharoperbu- 40.1 43.5 68.5 tylacetonicumQ97T12_Cacetobutylicum 40.9 40.4 100 (CaXynQ97)^(2,3)F1TBY8_Cpapyrosolvens 54.5 36.1 49.6 (CpXynC7I)² Q46961_Dehrysanthemi(EcXynA) 40.9 100 40.4 ¹Comparative analysis was performed with thesequence shuffling tool PRSS available athttp://fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=shuffle.²XynQ97, XynC7I, PpXyn30A and MpXyn30A are the four GH30-8 subsetxylanases being used to represent the disclosed GH30-8 xylanases of thepresent invention in this application and are included for comparativereasons. ³Sequnces with UniProt accession numbers F7ZYN8 and F0KEL6 arenot included as they are 100% identical to UniProt accession numberQ97TI2 and are therefore redundant.

Compositions Comprising the GH30-8 Xylanases of the Present Invention.

The present invention provides a composition comprising an effectiveamount of at least one GA-independent GH30-8 xylanase that is capable ofbreaking down lignocellulose material. The enzyme composition of theinvention may comprise a multi-enzyme blend, comprising more than oneenzymes or polypeptides of the present invention. The GH30-8 xylanasesubset composition of the invention can suitably include one or moreadditional enzymes derived from other microorganisms, plants, ororganisms. Synergistic enzyme combinations and related methods arecontemplated. One skilled in the art can readily identify the optimumratios of the GH30-8 enzymes to be included in the enzyme compositionsfor degrading various types of lignocellulosic materials to contributeto efficient conversion of various lignocellulosic substrates to theirconstituent fermentable sugars in the case of conversion of polymericsugars to monomers and to the desired oligomeric xylooligosaccharidemixture if that is desired. Assays known to the art may be used toidentify optimum proportions/relative weights of the GH30-8 xylanases inthe enzyme compositions, with which various lignocellulosic materialsare efficiently hydrolyzed or broken down in saccharification processes.

In one embodiment, the invention comprises a composition comprising aneffective amount of at least one of the novel GA-independent GH30-8xylanases of the present invention. By “effective amount” we mean anamount sufficient to catalyze or aid the digestion or conversion ofhemicellulose materials in lignocellulosic polysaccharide containingsubstrates to fermentable sugars or to a desired xylooligosaccharidecomposition and or to obtain a desired quality in the remaining xylancontaining materials providing the full or partial removal of xylan. Inone embodiment, an “effective amount” comprises the amount required toconvert polymeric xylan, under ideal conditions, to the limitxylooligosaccharides, with depletion of the polymer in a given period oftime. In one example, the combined weight of the novel GH30-8 xylanasesubset of the present invention having xylanase activity as measured byHPLC or biochemical reducing sugar assays can constitute about 0.05 wt.% to greater than 99 wt. % (e.g., about 0.05 wt. % to about 70 wt. %,about 0.1 wt. % to about 60 wt. %, about 1 wt. % to about 50 wt. %,about 10 wt. % to about 40 wt. %, about 20 wt. % to about 30 wt. %,about 2 wt. % to about 45 wt %, about 5 wt. % to about 40 wt. %, about10 wt. % to about 35 wt. %, about 2 wt. % to about 30 wt. %, about 5 wt.% to about 25 wt. %, about 5 wt. % to about 10 wt. %, about 9 wt. % toabout 15 wt. %, about 10 wt. % to about 20 wt. %, etc) of the totalproteins in the enzyme composition.

In one embodiment, the enzyme compositions desirably comprise mixturesof 2 or more, 3 or more, 4 or more, or even 5 or more GH30-8 xylanasesof the invention as defined above that can catalyze or aid the digestionor conversion of hemicellulose materials to a desired oligosaccharidemixture, their final xylooligosaccharide mixture or to fermentablesugars. It is expected that members of the GH30-8 subset, may functionsynergistically with xylanases of other enzyme families (including theGA-dependent GH30-8 xylanases), to more efficiently degrade xylan.Suitable xylanases include those having at least equal to or greaterthan 30% (e.g., at least about 30%, 35%, 40, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)identity to SEQ ID NOs: 1-4, over a region of at least about 10 (e.g.,at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,375, 400) residues.

The GH30-8 xylanase composition of the present invention may alsocomprise an effective amount of at least one of the novel GH30-8 enzymesof the present invention and at least a second additional enzyme havingenzymatic activity. For example, the composition may include enzymeshaving xylosidase activity, cellobiohydrolase activity, β-glucosidaseactivity, cellulase activity, β-xylosidase activity, arabinofuranosidaseactivity, lytic polysaccharide monooxygenase activity, lyase activity orendoglucanase activity.

The GH30-8 xylanase compositions of the present invention can suitablyfurther comprise one or more accessory proteins, such as, for exampleand without limitation, mannanases such as endomannanases,exomannanases, and 6-mannosidases; galactanases such as endo- andexo-galactanases; arabinases such as endo-arabinases and exo-arabinases;ligninases; amylases; α-glucuronidases; proteases; esterases such asferulic acid esterases, acetyl xylan esterases, coumaric acid esterasesor pectin methyl esterases; lipases; other glycoside hydrolases;xyloglucanases; CIP1; CIP2; swollenins; expansins; and cellulosedisrupting proteins such as cellulose binding modules; other xylanases,pectate lyases and arabinofuranosidases; stabilizers known to the art.Additives include, without limitation, any combination of sugars (e.g.maltose, glycerol), sugar alcohols (e.g. sorbitol), detergents (usuallynonionic) or thickeners and cryoprotectants (e.g. glycerol, propyleneglycol, polyethylene glycol).

The other enzymes or proteins of the composition of the currentinvention can be isolated or purified from a naturally-occurring source,or expressed or overexpressed by a recombinant host cell. They may beadded to an enzyme composition in an isolated or purified form. They maybe expressed or overexpressed by a host organism or host cell as part ofa culture mixture, for example a fermentation broth.

The GH30-8 enzyme compositions of the present invention are used or areuseful for producing metabolizable simple sugars in conjunction withother xylan accessory enzymes, such as α-glucuronidases,α-arabinofuranosidases, esterases and xylosidases. The GH30-8 subsetxylanases of the present invention, by themselves will generate xyloseonly as a nonspecific low rate side reaction of an already smallxylooligosaccharide. The xylooligomeric sugar mixture produced by theseenzymes can best be viewed in the TLC data of theQ97, C7I, PpXyn30A andMpXyn30A proteins (FIGS. 6, 10A-B and 20).

The GH30-8 enzyme compositions of the present invention are also used orare useful for reducing hemicellulosic polymers and cellulosic polymers(when synergistically applied with cellulase enzymes or lignocelluloseor cellulase disrupting proteins such as CBMs, expansins or swolleninsor lytic polysaccharide monooxygenases) into metabolizable carbonmoieties. The enzyme composition is suitably in the form of a product ofmanufacture, such as a formulation, and can take the physical form of aliquid or a solid.

Methods of Synthesis.

As described in the examples below, protein expression and purificationscheme is done as known in the art. The cells are grown in preparationof protein expression and during protein expression and are optimized asnecessary to increase protein yield. Such changes that might beconsidered for enhanced protein expression include: the specifictreatment of the cells during the inoculant growth, the specificconditions of the inoculation such as the quantity of cells used, thestarting amount of antibiotic selection, culture growth (and/or proteinexpression) temperature, the OD 600 nm measure when induction begins,whether IPTG or lactose is used to induce expression of the lacoperator, The concentration of the inductant, oxygen availability duringgrowth and more importantly during protein expression, and the length oftime provided for expression. The current systems of expression areprimarily based upon guidance provided by the pET System manual fromNovagen (10^(th) Edition) for use with IPTG/lactose inducible lacoperators in the Gram-negative bacterium Escherichia coli. The proteincoding region for these GH30-8 GA-independent xylanases of the presentinvention might also be expressed from any number of other inducibleexpression vectors or even nuninducible, leaky vectors. Other vectorsinclude expression that is inducible with other sugars such asarabinose, mannose or other chemicals or those that are responsive tophysical changes such as exposure to any wavelength of light,temperature change or chemical shock.

Using any of the protein expression systems described above, the methodsemployed for protein expression could also be altered to perform as anautoinduction system. Yields of these enzymes might also increasethrough the use of other protein expression hosts such as, but notlimited to other bacteria, yeast, fungi, plants and insects. Althoughnot expected to increased yields, these enzymes might also be producedthrough in-vitro synthesis or through purification of the desired enzymefrom the native source organism.

Methods of Use.

The GH30-8 xylanases and compositions of the present invention can beapplied in any industry for the reduction of xylans toxylooligosaccharides and novel mixtures of substitutedxylooligosaccharides or for the production of desired productcharacteristics resulting from the removal, partial removal, limiteddisruption or modification of xylan fraction. By “xylans” we mean aβ-1,4-linked xylose polysaccharide which is the primary hemicellulose ofhardwoods and crop residues and the second most abundant carbohydratepolymer in lignocellulosic biomass. Other forms of this hemicellulosicpolysaccharide can also be found in grain derived food products and infruit products. The source of the xylan polysaccharide typically definesits chemical characteristics in terms of chain length and sugar andnon-sugar substitutions along the xylan chain. The nature of thesubstitutions along the xylan chain define various xylan typesincluding, glucuronoxylan, acetylglucuronoxylan,acetylglucuronoarabinoxylan, glucuronoarabinoxylan and arabinoxylan, allgenerally referred to as “xylans”.

The GH30-8 xylanase and compositions thereof of the present inventioncan be used for hydrolyzing, breaking up, or disrupting all xylans orxylan-comprising compositions. In one embodiment, the method comprisescontacting the xylan or xylan-comprising composition with the GH30-8GA-independent subset of xylanases or enzyme composition of the presentinvention under suitable conditions, wherein the GH30-8 subset or enzymecomposition of the present invention hydrolyzes, breaks up or disruptsthe xylan or xylan-comprising composition.

The GH30-8 xylanases of the present invention and compositions thereofused in such a process may comprise, for example, a 0.1 g to 100 g(e.g., 2 g to 20 g, 3 g to 7 g, 1 g to 5 g, or 2 g to 5 g) ofpolypeptides having xylanase activity per kg of hemicellulose in thebiomass material. The GH30-8 xylanases of the present invention andcompositions thereof may be applied in conjunction with other enzymesfor complete enzymatic degradation of xylans to xylan-constituentmonosaccharides or by itself to produce complex xylooligosaccharidemixtures or otherwise facilitate the processing of the xylan fraction oflignocellulosics.

The GH30-8 xylanase and compositions of the present invention can alsobe used to digest xylans from any source, including all biologicalsources, such as plant biomasses, including, but not limited to, corn,grains, sugarcane, grasses (Indian grass, such as Sorghastrum nutans;or, switchgrass, e.g., Panicum species, such as Panicum virgatum),perennial canes (e.g., giant weeds), woods or wood processingbyproducts, e.g., in the wood processing, pulp and/or paper ornanocellulose and nanofibrilated cellulose industry, in textilemanufacturing, in household and industrial cleaning agents, and inbiomass waste processing; for the processing or preparation of dough orbread based products and in animal feed for application to enhanceanimal nutrition and feed digestion and possibly for the synthesis oflarger xylooligosaccharides as exemplified in Example 1.

The GH30-8 xylanases of the present invention and compositions thereof(including enzymes or designed enzyme compositions) can also comprise atleast one biomass material. By “biomass material” we mean any materialcomprising a lignocellulosic material derived from an agricultural cropor byproduct of a food or feed production. Suitable biomass material canalso include lignocellulosic waste products, waste paper or waste paperproducts, plant residues comprising grains, seeds, stems, leaves, hulls,husks, corncobs, corn stover, grasses, straw, reeds, wood, wood chips,wood pulp, or sawdust. Exemplary grasses include, without limitation,Indian grass or switchgrass. Exemplary reeds include, withoutlimitation, certain perennial canes such as giant reeds. Exemplary paperwaste include, without limitation, discarded or used photocopy paper,computer printer paper, notebook paper, notepad paper, typewriter paper,newspapers, magazines, cardboard and paper-based packaging materials.

The GH30-8 xylanase of the present invention and compositions thereof(including enzymes or designed enzyme compositions, such as products ofmanufacture or a formula) are useful for hydrolyzing hemicellulosicmaterials, catalyzing the enzymatic conversion of suitable biomasssubstrates to mixtures of complex oligomeric sugars or fermentablesimple sugars.

Methods of using or applying the GH30-8 xylanases and compositionsthereof in a research setting, an industrial setting, or in a commercialsetting are also provided. The GH30-8 xylanases of the present inventionand compositions thereof may be added as a desired mass of dry powder oras a desired volume of concentrated or diluted aqueous or non-aqueoussolution to xylan containing materials incubated at a desiredtemperature, which may include temperatures which are optimal or notoptimal (could be too hot (slow or fast inactivation throughdenaturation) or too cold (non-optimal activity)) for the GH30-8xylanases of the present invention. The application of the GH30-8xylanases of the present invention and compositions thereof may continueuntil such time as the desired outcome is achieved. For application asan additive to animal feed, the designated amount of GH30-8 xylanases ofthe present invention or compositions thereof will be added to, at thedesired proportion, a xylan containing biomass material or to anon-xylan containing material intended for animal consumption. Followingaddition, this animal feed material will be processed in a mannerconsistent with significant or acceptable GA-independent GH30-8 xylanaseactivity recovery for the anticipated application within the animalsdigestive tract.

It is also considered that these GH30-8 xylanases of the presentinvention and compositions thereof may be applied through surfacetreatments of biomass products and items for the alteration of woodsurface physical, chemical or textural properties. Using the GH30-8xylanases and compositions of the present invention to remove or reducexylans in biomass preferably yields 50% to 90% xylobiose and an array ofxylooligosaccharides of the enzyme accessible xylan.

In addition to reducing xylans in biomass to xylooligosaccharides andsugars, the GH30-8 xylanases and compositions of the present inventioncan be used in industrial, agricultural, human food and animal feed, aswell as a human food and animal feed supplementation. There is an everincreasing interest for the use of lignocellulosic biomass to makeproducts and fuels to increase our use of renewable resources and forreduction of greenhouse gas emissions. The GH30-8 xylanases andcompositions of the present invention may find applications in anylignocellulose based process in which the xylan component is treated forremoval, or for subsequent use in the form of xylooligosaccharides,substituted xylooligosaccharides, oligosaccharides of other polymericsugars or monosaccharides of any lignocellulose derived sugar. Forinstance, the GH30-8 xylanases and compositions of the present inventionmay find applications in wood, paper and pulp treatments, treatingfibers and textiles, treating foods and food processing, animal feedsupplementation and food or feed or food additives, reducing the massand volume of substantially untreated solid waste, detergent,disinfectant or cleanser (cleaning or cleansing) compositions. There isalso interest for use of such enzymes in processing of dough and in thepreparation of other foods and beverages and in the preparation ofprebiotics from both cereal grains and lignocellulosic biomass as foodadditives and nutraceuticals.

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description. As will be apparent, the inventionis capable of modifications in various obvious aspects, all withoutdeparting from the spirit and scope of the present invention.Accordingly, the detailed description of the novel compounds and methodsof the present invention are to be regarded as illustrative in natureand not restrictive.

III. Examples

The invention will be more fully understood upon consideration of thefollowing non-limiting Examples. The invention has been described inconnection with what are presently considered to be the most practicaland preferred embodiments. However, the present invention has beenpresented by way of illustration and is not intended to be limited tothe disclosed embodiments. Accordingly, those skilled in the art willrealize that the invention is intended to encompass all modificationsand alternative arrangements within the spirit and scope of theinvention as set forth in the appended claims.

Example 1 Structural and Biochemical Characterization CpXyn30A

In this example, we present the structural and biochemicalcharacterization of a novel enzyme that possesses a high degree of aminoacid identity to the canonical GH30-8 enzymes, but lacks the hallmarkβ8-α8 loop region which defines the GH30-8 subfamily of xylanases. Thethree-dimensional structure of this unique GH30 subfamily 8 homolog wasdetermined using x-ray crystallographic methods and provide functionalcharacterization of the enzyme with comparisons to the canonical GH30subfamily enzyme XynC from Bacillus subtilis (BsXynC).

In light of the role of the β8-α8 loop region in imparting functionalspecificity to the GH30-8 subfamily, amino acid sequence studies wereimplemented to identify homologs which possess sequence differences inthis region. A putative xylanase (UniProt ID: FITBY8; referred to asxylanase 30A) derived from the lignocellulose degrading bacteriumClostridium papyrosolvens (CpXyn30A) was identified and chosen forstudy.

DNA synthesis and Protein Expression.

The sequence for UniProt ID: C7IMC9 (also known as F1TBY8) wasidentified for this study and the expression-optimized coding sequence(Welch et al., 2009) including an C-terminal HisTag was ordered from DNA2.0 (Menlo Park, Calif.) in the kanamycin-resistant pJexpress 411expression vector. Plasmid DNA was used to transform Escherichia colifor expression. Cells were grown with shaking at 37° C. in Luria-Bertanibroth supplemented with 0.03 mg/mL kanamycin until they reached anoptical density at 600 nm of 0.6. The cells were induced by the additionof isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.5mM and incubated with shaking at 250 RPM for five hours at 37° C. Cellswere harvested by centrifugation at 7500 RPM for 20 minutes at 4° C. Theresulting pellet was suspended in 50 mM sodium phosphate, 100 mM NaCl,pH 7.2 at a ratio of 5 mL per gram of cell pellet. A 1 μL aliquot of 1×Halt protease inhibitor (Thermo Fisher, Rockford, Ill. USA) was addedfor every 1 mL of buffer used. Suspended cells were lysed usingsonication and the lysate was centrifuged at 11,000 RPM for 30 minutesat 4° C. The resulting supernatant was dialyzed overnight at 4° C.against 50 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, pH 7.2,using 10,000 Da MWCO dialysis tubing.

Purification.

The post dialysis material was centrifuged to remove any precipitate andthen filtered through a 0.22 μm filter to further remove debris. Thefiltered solution was then loaded onto a 1 mL HisTrap fast flow column(GE Healthcare Life Sciences Pittsburgh, Pa.) charged with Ni²⁺ and thehexahistidine-tagged recombinant protein was eluted with a lineargradient of 0-500 mM imidazole in 50 mM sodium phosphate, 250 mM NaCl,pH 7.2. Peak fractions were analyzed by SDS-PAGE (Laemmli, 1970).Fractions containing the protein were combined and dialyzed against 50mM sodium phosphate, 250 mM NaCl, 10 mM imidazole, pH 7.2, overnight at4° C. using 10,000 Da MWCO dialysis tubing. A second purification stepusing a 1 mL HisTrap column charged with Co²⁺ was then employed with thesame elution scheme as above. A single large peak was obtained from thisrun and peak fractions were analyzed by SDS-PAGE to ascertain purity andsize. Fractions containing the purified protein were combined anddialyzed into 20 mM HEPES, 150 mM NaCl, pH 7.2. In a separatepreparation meant solely for biochemical studies, CpXyn30A was purifiedas described, but the final purified protein was dialyzed against 30 mMTris HCl, 50 mM NaCl, pH 7.5. After dialysis, the protein solution wasconcentrated to at least 5 mg/mL and stored at −80° C. untilcrystallization or functional studies. The GH30-8 xylanase, BsXynC, waspurified as previously described (St. John et al., 2011; St. John etal., 2006). Protein concentration was routinely determined usingabsorbance at 280 nm with the ProtParam predicted extinction coefficient(Gasteiger et al., 2005).

Crystallization.

The sparse matrix screens Crystal Screen, Crystal Screen 2, Index andPEG/Ion as well as the Sodium Malonate, and Ammonium Sulfate GridScreens (Hampton Research, Aliso Viejo, Calif.) were used for theinitial screening. Sitting-drop vapor-diffusion experiments wereperformed in 24 well microplates (Art Robbins Instruments, Sunnyvale,Calif.). Each well contained 300 μL of precipitant solution and dropswere set using 1 μL of protein solution and 1 μL of precipitant. Theplates were sealed with sealing film and incubated at 25° C. Acrystallization condition of 0.1 M ammonium acetate, 0.1M Bis-Tris pH5.5 and 17% polyethylene glycol 10,000 was found using the HamptonResearch Index screen. Sitting-drop vapor-diffusion experiments wereperformed using this condition at protein concentrations of 6.5 mg/mLand 10 mg/mL and cubic crystals (˜0.5 mm on edge) were obtained in thedrops containing CpXyn30A at a concentration of 6.5 mg/mL. Crystals wereharvested for cryocrystallographic data collection by transferring themstepwise to solutions containing 5% (v/v), 10% (v/v) and 20% (v/v)glycerol in well solution. After the 20% glycerol transfer, the crystalswere flash cooled and stored in liquid nitrogen until data collection.

Data Collection, Analysis and Model Building.

Data was collected on a Rigaku RU-H3R copper rotating anode generator,operating at 50 kV and 100 mA, fitted with Confocal Maxflux™ optics(Osmic Inc., Troy, Mich.) and a Rigaku R-Axis IV+ image plate detector.A 180° dataset was collected with 5 minute exposure times and a Phioscillation of 0.5 degrees per image. The resulting data was processedto 2.01 Å and the crystal belonged to the orthorhombic space group,C222₁ with unit cell parameters: a=66.0, b=76.5, c=150.6 anda=β=γ=90.00. Data were indexed and integrated in iMosflm (Battye et al.,2011), scaled in SCALA (Evans, 2005), and initial molecular replacementphases, electron density map calculation and model building wasperformed with the programs Phaser (McCoy et al., 2007), Phenix (Adamset al., 2010) and Coot (Emsley et al., 2010), respectively. The finalmodel (PDB code:4FMV) was studied and figures prepared using PyMOL(DeLano, 2002).

Biochemicals and Assays.

All reagents were of the highest purity available. Xylooligosaccharidesxylobiose (X2) and xylotriose (X3) were purchased from WAKO Chemicals(Richmond, Va.) and xylotetraose (X4), xylopentaose (X5) and xylohexaose(X6) were purchased from Megazyme International (Wicklow, Ireland).Concentrations of xylooligosaccharide standards were determined with thephenol-sulfuric total carbohydrate assay (Dubois et al., 1956). Thealdouronate, aldopenturonate (GX4), with a GA residue substitutedpenultimate to the nonreducing terminus of xylotetraose was thealdouronate limit product of a GH11 xylanase (Biely et al, 1997)(Trichoderma longibrachiatum, XynII, Hampton Research, Aliso Viejo,Calif.) and was purified using a 1.7 m P-2 resin column (Bio-Rad,Hercules Calif.) in 50 mM formic acid. The Rotovap concentrated sugarwas then loaded onto the same column equilibrated with water to removethe formic acid from the oligosaccharide. The desalted GX4 waslyophilized, dissolved in water and the concentration determined withthe Blumenkrantz assay for total uronic acid content (Blumenkrantz &Asboe-Hansen, 1973).

Enzymatic Activity Measurements and Hydrolysis Product Studies.

Activity measurements on polymeric substrates were determined throughreducing end quantification with the Nelson's Test (Nelson, 1944) as hasbeen previously described (St. John et al., 2006). Conditions forhydrolysis by CpXyn30A were optimized using beech wood xylan(Sigma-Aldrich Corporation St. Louis, Mo.) in acetate buffers ranging inpH from 3-6. Thermostability was analyzed using enzyme pre-incubationsat a range of temperatures from 4°-50° C. followed by activityassessment at 30° C. Activity measurements for functional comparisonwere performed using sweet gum wood glucuronoxylan (SGX) (kindlyprovided by James F. Preston from the University of Florida) and wheatarabinoxylan (WAX) (Megazyme International). Hydrolysis ofxylooligosaccharides was determined using an Agilent 1260 HPLC (AgilentTechnologies, Santa Clara, Calif.) with resolution of neutralxylooligosaccharides performed using a Phenomenex RNO column (PhenomenexTorrance, Calif. USA) with water as eluent at 0.3 ml/min flow and 75° C.or a Shodex SH1821 (Showa Denko America, New York, N.Y.) in 0.05% H₂S0₄running at 0.8 ml/min and 75° C. In both cases, the refractive index ofthe eluate was monitored throughout the separations.

Hydrolysis reactions were performed in 25 μL volumes under optimizedconditions (100 mM sodium acetate, pH 4.5, at 30° C.) withxylooligosaccharides at 12.1 mM; a concentration (of X6) thatapproximated that of the polysaccharide used in the reactions employingpolymeric xylan substrates. Reactions were stopped by boiling thesamples in a water bath for 5 minutes. For studies evaluationp-nitrophenol (pNP) conjugated xylooligosaccharides (pNPXn, where n=thenumber of xylose units) samples were injected onto an Agilent 1260 HPLC(Agilent Technologies, Santa Clara, Calif.) equipped with a Zorbax C8column (Agilent Technologies) and were eluted with a 0-90% acetonitrilegradient in water (Eneyskaya et al., 2003) and absorbance of the eluatewas monitored at 302 nm. All HPLC analyses were performed in triplicateusing 5 μL injections. Thin layer chromatography (TLC) was performed asdescribed previously (St. John et al., 2006; Bounias, 1980).

Amino Acid Sequence Studies.

Sequences were aligned with the program MAFFT (Katoh & Toh, 2008) andthe alignment figure was generated with ESPript (Gouet et al., 2003).Domain prediction was done using the online Conserved Domains program(Marchler-Bauer et al., 2011) and the domain representation was createdusing the program DOG 1.0 (Ren et al., 2009). Phylogenetic relationshipswere calculated and represented using the software MEGA 6.0 (Tamura etal., 2013).

Selection of CpXyn30A.

Primary amino acid sequence alignments (FIG. 7 a) identified a uniqueGH30-8 enzyme that did not contain the normally conserved β8-α8 loopsequence. For this enzyme, (UniProt ID: F1TBY8) sequence studiesverified the existence of the two conserved glutamate amino acid sidechains which catalyze the double displacement reaction common to CAZyClan A enzymes and identified a likely secretion signal sequence inaddition to non-catalytic modules positioned C-terminal of the GH30-8catalytic module. These include a family 6 carbohydrate binding module(CBM6) for binding soluble glucan and two dockerin domains presumablyfor interaction within a cellulosome assembly (FIG. 7 b). Combined,these features suggest that this enzyme may have a role in thedegradation of the xylan component of lignocellulosic biomass.Phylogenetic analysis of CpXyn30A verifies the most similar enzymes tobe GH30-8 homologs from Gram positive organisms (FIG. 7 c).

Based on these findings and an interest in characterizing a GH30-8enzyme with a nonconserved amino acid sequence in the β8-α8 loop, thecoding sequence for the gene was ordered from DNA 2.0 with the Signal-P(Petersen et al., 2011) predicted secretion signal sequence replaced byan amino-terminal methionine and a hexahistidine tag appended to the newcarboxy-teminus defined by the end of a GH30-8 sequence alignmenteffectively truncating the protein sequence before the predicted(Bateman et al., 2004) CBM6 and dockerin domains.

Structure of CpXyn30A.

Refinement and model quality statistics for CpXyn30A structure model arepresented in Table 2.

TABLE 2 Data collection and refinement statistics of Clostridiumpapyrosolvens Xyn30A. PDB code 4FMV Wavelength (Å) 1.542 Resolutionrange (Å) 38.30-2.01 (2.08-2.01) Space group C2221 Unit cell parametersa, b, c (Å) 66.0, 76.5, 150.6 Total reflections 46801 (4031) Uniquereflections 24506 (2121) Redundancy 1.9 (1.9) Completeness (%) 94.9(85.7) Mean I/sigmaI 27.63 (12.32) Wilson B-factor (Å²) 16.05 R-merge0.020 (0.058) R-meas 0.028 CC* 1.00 (0.996) R-work 0.1642 (0.1534)R-free 0.2010 (0.2341) CC(work) 0.949 (0.955) CC(free) 0.928 (0.708)Number of atoms 3228 Ligands 0 Waters 212 Protein residues 386 RMS(bonds, Å) 0.013 RMS (angles, °) 1.50 Ramachandran favored (%) 97Ramachandran outliers (%) 0 Clash score 2.71 Average B-factor (Å²) 15.20Solvent (%) 18.10 Statistics for the highest-resolution shell are shownin parentheses.

As expected, the overall structure of CpXyn30A is very similar to otherGH30-8 enzymes with an RMSD of just 0.95 Å (all-atoms, Pymol: align)obtained when compared to the crystallographic structure of thecanonical Gram-posative GH30 xylanase, BsXynC (FIG. 8 a). However, thestructure of CpXyn30A in the β8/α8 catalytic core domain is notablydifferent from BsXynC (PDB code: 3KL5) in the β1-α1, β2-α2 (FIGS. 8 b &8 c), and, most importantly in the CpXyn30A unique β7-α7 and β8-α8 loopregions (FIGS. 9 a & 9 b).

In the β1-α1 loop region, the sequence of CpXyn30A is shorter by threeamino acids compared to BsXynC and the Gram-negative bacterial GH30-8enzyme, XynA, from Erwinia cluysanthemi (EcXynA). A conserved tryptophanresidue (Trp25) positioned by this loop establishes a predicted −3xylosyl binding subsite (St. John et al., 2011; Urbanikova et al.,2011). While the Cα position for this conserved tryptophan is nearlyidentical in both BsXynC and EcXynA, the position of Trp25 in CpXyn30Ais shifted 3.4 Å towards the inside portion of the loop. In either case,the indole side chain of the tryptophan lies in a similar position inall three enzymes (FIG. 17). As expected it is unique in the β7-α7 andβ8-α8 loop regions relative to BsXynC and also, as expected, uniquecompared to CpXyn30A

The β2-α2 loops of BsXynC and EcXynA are both very similar but theanalogous region of CpXyn30A is considerably larger due to the presenceof an additional nine amino acids in the α2 helix. In all three enzymes,a single aromatic amino acid stacking interaction is observed betweenresidues in helices α2 and α3 (FIG. 8 b). The conserved interactionshared by these three xylanases consists of a phenylalanine (BsXynC andEcXynA) or tyrosine (Tyr124 in CpXyn30A) from the α3-helix in aperpendicular stacking arrangement with a tryptophan (Trp66 in CpXyn30A)extending from the α2-helix. The extendedα2 helix unique to CpXyn30Aprovides two additional intramolecular contacts with adjacent regions ofthe enzyme.

In the first interaction, Trp58 of CpXyn30A overlays the α3-helix andstacks perpendicular to Tyr120 (FIG. 8 b). The second interaction foundin the extended loop region is a hydrogen bond between Asp56 and Ser93of the β4-α4 loop region. These last two contacts are not found in theBsXynC or EcXynA enzymes and may serve a role in supporting thebeta-structured β3-α3 loop region as originally described in BsXynC (StJohn et al., 2011), but not in EcXynA.

A structural difference between GH30-8 enzymes from Gram-negativebacteria and those from Gram-positive bacteria (St. John et al, 2011) isfound in the β3-α3 and β4-α4 loop regions (FIG. 8 c). In CpXyn30A, thisregion adopts a fold similar to the Gram-positive GH30-8 enzymes(BsXynC-like) with a small β-structure extending upward at the top ofthe β3-α3 loop and a region in the β4-α4 loop which supports thisextended β-structure primarily through stacking interactions. This is incontrast to the Gram-negative homologs of these enzymes (EcXynA like)which lack the extended β3-α3 loop β-structure and instead relies solelyon hydrogen bonding between the two loop regions for stabilizingcontacts. These hydrogen bonds are not present in the Gram-positiveexamples of these enzymes (St. John et al., 2011).

The β7-α7 loop region (FIG. 9) of CpXyn30A also displays a significantlydifferent structure relative to the BsXynC and EcXynA enzymes. In thisloop region of BsXynC and EcXynA, two conserved amino acids (Tyr231 andSer235 in BsXynC) establish hydrogen bonds with the C-2 and C-3 hydroxylgroups of the α-1,2-linked GA appended on the xylan chain (FIG. 9 a).

In CpXyn30A, this loop is smaller in size, but still has the conservedtyrosine (Tyr234). Following this amino acid, the loop region divergesslightly from the typical structure with Asp236 in place of a normallyconserved serine and is positioned as to make a functionally similarcontact unlikely. Despite this difference, it may be considered possiblethat the O-2 hydroxyl of sugars linked α-1,2 to the xylose in thissubsite (typically GA) may hydrogen bond with Tyr234 as observed in theligand bound crystal structures of BsXynC (FIG. 3 a) and EcXynA (St.John et al., 2011, 291; Urbanikova et al., 2011).

In the altered sequence of the β8-α8 loop, four of the GA coordinatingcontacts identified for the ligand bound BsXynC structure are no longeravailable (FIG. 3 b) (St. John et al., 2011). Surprisingly, despite thefact that the sequence of the β8-α8 loop region of CpXyn30A iscompletely different from the conserved sequence found in BsXynC andEcXynA, the structure of the loop does not significantly deviate fromthe Ca-trace of these model enzymes. This is most noteworthy since thisregion forms the basis for classification of the proteins into theGH30-8 subfamily due to its importance in GA recognition (FIG. 3 b).

Functional Characterization.

While CpXyn30A has measurable activity on glucuronoxylan, the specificactivity is low relative to the characterized GH30-8 xylanases as wellas other more common β-1,4-endoxylanases such as those from familiesGH10 and GH11. In consideration of this finding, other polymericsubstrates were tested for activity. These includedcarboxymethylcellulose, barley β-glucan, yeast glucan, glucomannan,galactoglucomannan, xyloglucan and gum arabic, but in each case therewas no detectable activity. The results presented in Table 3 indicatethat CpXyn30A displays similarly low specific activity on all xylansubstrates tested.

TABLE 3 Specific activity¹ comparison of CpXyn30A and BsXynC on xylansand xylooligosaccharides. Substrates Concentration CpXyn30A BsXynCSweetgum 10.00 mg/ml 1.1 ± 0.1  70.7 ± 4.8 glucuron- 7.50 mg/ml 1.1 ±≦0.1 61.7 ± 3.8 oxylan (SGX) Wheat 7.50 mg/ml 1.7 ± 0.2  nd²arabinoxylan (WAX) Xylo- 12.10 mM 1.19 ± ≦0.01     0.019 ± ≦0.002⁵hexaose (X₆)⁴ Xylo- 12.10 mM 0.36 ± ≦0.01 ND³ pentaose (X₅)⁴ ¹Units/mgprotein, where one Unit is defined as one μmole/minute of activity. Dataresults from triplicate measurements resulting from a single assay.These results were consistent with numerous previous analyses. The givenerror is represented by the standard deviation. ²nd = Not detected ³ND =Not determined ⁴The data for these substrates represent an evaluation ofspecific activity based solely on the decrease of substrate. Thesevalues are higher than the true specific activity as the describedcompeting transglycosylation reaction presumable consumes two X₆molecules. Xylohexaose was digested for 8 minutes and xylopentaose wasdigested for 20 minutes. ⁵The X₆ substrate concentration was only 10 mMfor this reaction, a difference in the comparison which is consideredinconsequential to this study.

Interestingly, specific activity was 57% greater on WAX than on SGX whenmeasured at the same substrate concentration (7.5 mg/ml). Studiesemploying X6 as a substrate at 12.1 mM (roughly the molar equivalence of10 mg/ml xylan) show that CpXyn30A exhibits a similar activity as with10 mg/ml SGX a characteristic not previously observed for other GH30-8enzymes (see below).

In TLC analysis of an overnight hydrolysate of SGX by CpXyn30A, X2, X3,X4 and the primary aldouronate, GX4 (aldopentauronate, FIG. 10A) wereobserved. However, without further studies, the configuration (i.e. GAsubstitution position) of this aldouronate product is unknown.

CpXyn30A also efficiently processed WAX with only low levels of X2 andX3 apparent following an overnight digestion, but numerous other spotswere observed on the plate which did not align with any of ourstandards. This suggests they are arabinofuranose substitutedxylooligosaccharides instead of neutral oligoxylosides. Hydrolysis of X6and X5 resulted in a distribution of smaller xylooligosaccharidessimilar to those observed for glucuronoxylan hydrolysis. There was onlyslight hydrolysis of X4 observed (FIG. 10B) and no detectable hydrolysisof X3 in overnight reactions (FIG. 10B).

Activity measurements of BsXynC confirm the reported function of thisenzyme as a glucuronoxylan xylanohydrolase which requires a substitutionof α-1,2-linked GA residues for activity. Multiple attempts were made toobtain activity measurements for the hydrolysis of WAX by BsXynC,including one attempt which used a 10-fold greater amount of enzyme thanthat used in similar reactions employing CpXyn30A and an overnightreaction time. However, all results were generally too variable andclose to zero to be reported as anything other than ‘not detected’(Table 3).

In agreement with previous findings, it is observed that BsXynC activityon X6 (10 mM) was 3-orders of magnitude (6172 fold) lower than on SGX(at 7.5 mg/ml) (Urbanikova et al., 2011), supporting the requirement forthe GA appendage for activity.

Parallel TLC studies of the reaction products generated by BsXynC alsoconfirm our current understanding of these enzymes and have providedfurther insight to their specificity (FIGS. 10A & 10B). Hydrolysis ofSGX by BsXynC yielded an array of aldouronate sugars (St. John et al.,2006; Vrsanska et al., 2007) while reactions containing WAX as substratedid not yield any detectable smaller sugars, a result supported by thelack of detectable enzymatic activity on this substrate in kineticstudies (Table 3). The TLC analysis of BsXynC hydrolysis of X6 visuallyconfirms the results presented in Table 3 with a very low activityobserved for hydrolysis of this substrate. In contrast to theseobservations, overnight hydrolysis of GX4 by BsXynC resulted in xyloseand the smaller aldouronate, aldotetrauronate (FIG. 10B).

Xylooligosaccharide hydrolysis studies showed that CpXyn30A has acompeting transglycosylase activity (Shaikh & Withers, 2008). For thisto occur, following the nucleophilic attack on the anomeric carbon bythe catalytic nucleophile (E232 in CpXyn30A) an enzyme-substrate complexis formed which, in retaining glycosyl hydrolase enzymes (such asCpXyn30A), typically resolves by release of the sugar from the enzymethrough a water molecule activated by the other member of the catalyticacid/base pair (E143 in CpXyn30A) (Shaikh & Withers, 2008). Thisaccepted, double-displacement reaction scheme generates smaller sugarsfrom polymeric substrates. However, if a sugar molecule were to bindinto the active site cleft instead of a water molecule, then the C-4hydroxyl group of the non-reducing terminal residue may becomeactivated, resulting in a transglycosylation reaction creating a newβ-1,4-xylosidic bond instead. For retaining glycosyl hydrolases likeGH30 enzymes, transglycosylation may occur as a product of a failedhydrolytic reaction.

Here, reaction mixtures consisting of X6 as the most abundant substratewere employed to probe the transglycosylase activity of CpXyn30A. If X6binds so that it will be hydrolyzed into two molecules of X3, and asecond X6 reoccupies the other half of the active site cleft, then thecondensation of these two sugars will result in the formation of thexylooligosaccharide xylononaose (X9) (FIG. 11, inset). Because aninitial hydrolytic activity is required to observe transglycosylation,the net reaction proceeds toward the right (smallerxylooligosaccharides) due to the eventual buildup of limit productswhich do not act as substrates for further endo-hydrolysis.

In the present study, hydrolysis of X6 by CpXyn30A results in what ispredicted from HPLC chromatograms as xylodecaose (X10) and X9 as well assmaller xylooligosaccharides such as X2 through X4 (FIGS. 11 & 12).These data were confirmed by TLC analysis which showed that within 20minutes of the start of the reaction, hydrolysis of X6 resulted in aspot with no mobility (est. DP degree of polymerization >8) and smallamounts of X4, X3 and X2. Formation of X10 and X9 may occur through atransglycosylation when CpXyn30A cleaves X6 such that either a X3 or X4is positioned in the glycone side of the substrate binding cleft in theenzyme substrate complex. Because xylooligosaccharides smaller than X5are not hydrolyzed they cannot be a source for furthertransglycosylation.

Based on this analysis, specific activities (Table 3) are anticipated tobe lower than reported as the enzyme catalyzed transglycosylationreaction consumes two molecules of X6. It is impossible to determinewhat the ratio of hydrolysis:transglycosylation reactions might bewithout quantification of X9 and X10.

Transglycosylation does not likely contribute substantially duringinitial hydrolysis of polymeric xylan as the concentration of reducingtermini is much lower and unlikely to significantly compete as anacceptor through the limited reaction time. Interestingly, even thoughthe model enzyme BsXynC only hydrolyzes neutral xylooligosaccharidessuch as X6 very slowly, the activity that was observed appears from TLCto resolve in part by transglycosylation, similar to CpXyn30A (FIG. 4b). Similar results were previously reported for another GH30-8Gram-positive enzyme (a highly conserved homolog from Bacillus sp.Strain BP-7) (Gallardo et al., 2010).

Our data on the rate of hydrolysis of X6 agrees with the previouslyreported level of activity of the Gram-negative GH30-8 enzymes EcXynAhaving 3-orders lower activity on this neutral xylooligosaccharide thanon a polymeric glucuronoxylan substrate (Urbanikova et al., 2011).Transglycosylation can also be observed by TLC after the ovrnightdigestion of GX. This indicates that BsXynC may be producingdisubstituted aldouronates (FIG. 4B).

Clues as to the distinctive comparative function of these enzymes may beascertained from their structures. Of the five hydrogen bonds and onesalt bridge that have been described which establish the interactionbetween the β7-α7 and β8-α8 loops and the GA side chain in the BsXynCand EcXynA enzymes, only two hydrogen bonds are thought to still bepossible in CpXyn30A. These positions, equivalent to Tyr234 and Trp265in CpXyn30A may be available for hydrogen bonding with either GA orarabinofuranose substitutions linked α-1,2 on the main xylan chain.However, since activity measurements are similar on neutralxylooligosaccharides and xylans, it seems unlikely that this potentialhydrogen bonding position plays a significant role in xylan hydrolysis.Instead, the β8-α8 loop contains larger, hydrophobic amino acids whichwould appear from inspection of a surface analysis to displace thexylose and any substitution in this position (−2 subsite). Because ofthis displacement, substitutions in this region most likely are beyondhydrogen bonding distance of Tyr234 and Trp265. The increased size ofthe β8-α8 loop may reorient the glycone bound xylan sugar out of anideal orientation for hydrolysis.

From these data, CpXyn30A stands out as a defunct GH30-8 xylanase havingno apparent specificity for O-2 linked GA substitutions and a greatlydecreased specific activity on the usual glucuronoxylan substrate whilesimultaneously possessing a unique ability to hydrolyze WAX, SGX and theneutral xylooligosaccharide X6 at rates approximately 100-fold greaterthan BsXynC processing of neutral sugars. Even though CpXyn30A has ademonstrated xylanase activity, it is not known whether the enzymerepresents an evolved functionality whose role has not yet beenidentified or a residual xylanase activity resulting from unbeneficialchanges to the Xyn30A gene in C. papyrosolvans.

The data presented helps us understand the function of the GH30-8 β8-α8loop in determination of the specificity of these enzymes. It seemsclear that the conserved sequence of this loop found in theBsXynC/EcXynA enzymes may not only enable recognition of theα-1,2-linked GA appendage, but might also prevent binding of neutralsugars by physically obstructing access to the binding cleft.

Example 2 Expression and Purification of CaXynQ97

The codon optimized (for E. coli) coding sequence for CaXynQ97 includinga C-terminal His-tag was synthesized by DNA 2.0 and inserted into theirpJexpress 411 kanamycin selective expression vector (pCaXyn30A).Chemically competent E. coli BL21 (DE3) was transformed with thepCaXynQ97 expression vector and selected for on LB agar platescontaining 50 ug/ml kanamycin. The following day a single colony wasselected from the plate and inoculated into a 50 ml volume of LB mediacontaining 50 ug/ml kanamycin contained in a 250 ml long-neck shakeflask. This was grown overnight at 37° C. with shaking at 250 rpm. Thefollowing morning, 5 ml aliquots of this culture were used to inoculateseveral 37° C. preequilibarted 0.5 liter volumes of LB media containing50 ug/ml kanamycin contained in a Fernbach flask. This was grown at 37°C. with shaking at 300 rpm until a measured OD 600 nm of approximately0.7 and the culture was then induced by addition of IPTG to a finalconcentration of 1 mM. The induced culture was then grown for anadditional 4-5 hours at 37° C. and shaking at 300 rpm. Followinginduction, the foil cap was kept in place for 1 hour, but removed forthe remaining hours of induction. The cells were then collected throughcentrifugation at 10400×g (i.e., 8000 rpm in a GSA rotor) at 4° C. Eachpellet resulted from 0.5 liter of expression culture. The pelleted cellswere stored frozen at −80° C. until used for protein purification.

The His-tagged version of this enzyme was purified in a standard mannervery similar to CpXyn30A (C7I) with use of a Ni-affinity IMACchromatography column and subsequent gel filtration chromatography.After several years attempting to obtain protein crystals forcrystallographic structure determination, we decided to redone theCaXynQ97 enzyme from the pJexpress 411 vector into the pET28 proteinexpression (Novagen) with removal of the C-terminal His-tag. The newpET28 based expression construct (pCaXynQ97-nohis) transformed into E.coli BL21 (DE3) expressed very well as did the previous pJexpressconstruct. The preceding expression protocol and the following cellprocessing procedure apply to both constructs except that for the cellprocessing for the no-his-tag expression product was “beefed-up” withaddition of lysozyme in the hope that it would make for a cleanerpreparation since no affinity tag was being used. As will be explainedit turned out that this purification was actually just as easy as anaffinity system due to the inherent high isoelectric point of CaXyn30A.

For protein purification, four pCaXynQ97-nohis E. coli proteinexpression pellets were thawed at room temperature and then on ice. AnEDTA-free Mini cOmplete protease inhibitor tablet (Roche) was added toone of these pellets. A volume of 8 ml of 25 mM Tris HCl pH 7.1 wasadded to each pellet and the soft pellets were resuspended using a glassrod and eventually, to obtain fluidity, with the action of α5 ml pipet.Resuspended pellets were combined and each of the four centrifuge tubes(250 ml volume) were rinsed with 2 ml of Tris buffer. Lysozyme was addedto a final concentration of 20 ug/ml and the full volume (˜47.5 ml) wastransferred to a 250 ml capacity glass sonication vial and allowed tocool on ice for 15 minutes. The sonic microtip was calibrated accordingto instructions and then submerged ˜1 inch below cell suspensionsurface. Set to 20% power (˜95 watts) and an approximate control knobsetting of 3.5. Cycle twelve times of 10 seconds on, 50 seconds off inice/water. Total process time is 12 minutes. Collect sonicate, and basedon approximate volume add 1M MgCl₂ to a final concentration of 2 mM andlysozyme as added before. Add 250 Units of Benzonase (Novagen) and rockat room temperature for 30 minutes. Cell lysate volume is split into2-45 ml Oakridge centrifuge tubes. Preequilibrate centrifuge to 15° C.and centrifuge cell lysates for 30 minutes at 15° C. at G-Force (i.e.;14000 rpm (SS-34 rotor)). Collect supernatant cell free extract andfilter through 0.45 um syringe tip filter.

The processing of both the His-Tag and no-His-Tag versions of CaXynQ97used Tris based buffers as it was shown several times that this enzymeprecipitates in the phosphate based buffers typically used for IMACchromatography, as in the purification of CpXynC7I. The resulting cellfree extract (CFE) of the CaXynQ97-nohis enzyme was fractionated on a 5ml Econo-Pac CM column (Bio-Rad) equilibrated in pH 7.1 25 mM Tris HCland a gradient to 500 mM NaCl. The fractionation was very clean and theeluted protein peaks were combined and concentrated using an AmiconUltra 15 with 10K MWCO. This concentrated CaXynQ97 preparation was thendesalted using a 5 ml Econo-Pac P-6 desalting column and then againconcentrated with a another Amicon Ultra 15 10K MWCO centrifugalconcentrator. It was noted that high concentrations of CaXynQ97-nohisdid not seem to like the high salt, so prior to concentrating forsubsequent desalting the enzyme was diluted with the Tris. Thepreparation was then purified on a Superdex 200PG 16/600 column (GEhealthcare) equilibrated with 25 mM Tris HCl pH 7.5, 100 mM NaCl.Concentrated and buffer exchanged into 25 mM Tris HCl pH 7.1. RemainingNaCl is estimated at <5 mM. This form of CaXynQ97-nohis proved to bevery soluble remaining is solution at concentrations greater than 100mg/ml. Protein crystal screening was performed with a preparation at ˜40mg/ml.

Functional characterization.

Wheat arabinoxylan was digested with the same number of Units ofactivity of the three enzymes tested. Following a pre-established timethe reaction was killed by heating to 90° C. for 10 minutes. Thereaction volume was then adjusted to 90% ethanol by addition of 100%ethanol and allowed to precipitate overnight at 4° C. The resultingmaterial was centrifuged at 20000×g for 30 minutes at 4° C. and thesupernatant and the pellet were isolated. The pellet was washed withcold 100% ethanol and the supernatent was rotovaped to remove all theethanol and then brought to a known volume. The pellet was resuspendedto this same volume. Total reducing end was determined with the Nelson'stest and total carbohydrate was determined with the Phenol Sulfuricassay. The degree of polymerization (DP) was calculated as the quotientof these two values and used to characterize the oligomeric state andhence information regarding the xylanases used.

Comparison of XynQ97 and XynC71.

Representative examples of the disclosed GH30-8 enzymes (XynQ97 andXynC71) differ slightly in their respective hydrolysis product profiles,but it is clear they are very similar when compared to the canonicalGH30-8 enzyme family homolog XynC. They are shown to yield a series ofsmall xylooligosaccharides and aldouronates indicating the enzymes haveno obvious preference for the GA xylan chain appendage. This is furthersupported by the arabinoxylan limit hydrolysis by these enzymes, incomparison to XynC, which was unable to degrade this substrate in anyway (FIG. 6 and FIG. 10A), the enzymes of the present invention readilyprocessed this substrate to small xylooligosaccharides and smallarbinofuranose substituted xylooligosaccharides.

Notably, based on the intensity of the remaining sample spot for theTLCs of FIG. 6 and FIG. 20 (bottom spots), the disclosed enzymesprocessed the arabinoxylan to smaller oligoxylosides and arabinofuranosexylooligosaccharides more efficiently than the GH10 xylanase XynA1CDindicating that the disclosed enzymes are better at cleaving xylanchains in regions of frequent side chain substitution.

XynC71, while it has a comparably low rate of hydrolysis (see Example 1)also appears to process wheat arabinoxylan just as efficiently asXynQ97, but glucuronoxylans to a slightly lesser degree (FIG. 6).Relative to the limit hydrolysis product profile of a typical GH10xylanase and a canonical GH30-8 appendage specific xylanase on either ofthese two xylans types, XynQ97 and XynC71 act in a similar fashion dueto the altered β7-α7 and β8-α8 loops.

Our data suggests that the GH30-8 xylanases of the present invention areable to hydrolyze highly substituted xylan substrates where otherxylanases are unable to function due to steric interaction between theenzyme and xylan side chain substitutions, thereby liberating moresubstituted xylooligomers and yielding more oligosaccharide sugarliberated from highly substituted often insoluble xylan substrates(Table 4).

TABLE 4 Comparison of XynQ97 with Industry Leading GHl0 Xylanase. TotalAnal- Supernatant/ Total RE Carbohydrate Enzyme ysis Pellet (umoles)(umoles) DP XynQ97 1 Supernatant 32.87 218.15 6.63 Pellet 0.191 5.1427.1 2 Supernatant 27.00 191.69 7.1 Pellet 0.298 5.26 17.7 Industry 1Supernatant 39.48 230.9 5.84 Leading Pellet 0.232 9.92 42.84 GH10 1Supernatant 39.19 184.1 4.69 Xylanase Pellet 0.750 18.78 21.12PbXyn10A1CD 2 Supernatant 28.6 175.81 6.14 Pellet 1.53 36.01 23.57

Example 3 Methods of Using the GH30-8 Xyalanases to Reduce Biomass

The enzymes and enzyme compositions of the present invention can be usedto hydrolyze biomass materials or other suitable xylan containingfeedstocks.

The GH30-8 xylanases and compositions thereof provided above are usefulin reducing xylans found in any source, including biomass, due to theirunique structure. Specifically, without the canonical GH30-8 subfamilyspecific loop regions which define the GA appendage specificity of theseenzymes, the GH30-8 subset of enzymes presently disclosed are consideredfree of such restrictions or limitations and therefore appears tofunction more generally as a β-1,4-endoxylanase, not having clearpreference toward xylan side chain appendages. These GH30-8 xylanases ofthe present invention are therefore more comparable to the very commonβ-1,4-endoxylanases of glycoside hydrolase families 10 and 11 whilesimultaneously being unique from these by yielding unique hydrolysisproduct profiles and obtaining better liberation of oligomers fromhighly substituted xylans.

These “generic” GH10 and GH11 endo-β-1,4-xylanases process xylans byworking around the appendages like obstacles which prevent the enzymefrom accessing the xylan chain. In doing this the GH10 endoxylanase isknown to produce the smallest of the GA-substituted xylooligosaccharides(aldotetrauronate). This is because the substrate binding cleft of thesexylanases can accommodate 2-GA appendages separated by just two xyloses(−3 and +1 subsites).

Using the canonical GH30-8 xylanase as a comparison it is known thatthese enzymes specifically bind the GA in the −2 subsite (FIGS. 2 & 3).Further, the limit hydrolysis product analysis yields the smallest GAsubstituted xylooligossacharide as aldotriuronate (see FIG. 6) resultingfrom a digest of beechwood glucuronoxylan by XynC). This indicates thatthe GH30-8 xylanases of the present invention appear likely toaccommodate 2 GA appendages separated by just one xylose.

This is supported by the limit hydrolysis of both beechwood xylan andwheat arabinoxylan by XynQ97 (FIG. 6). For the enzymes representing theGH30-8 and GH10 xylanase families, the limit products show the expectedresults from many previously reported biochemical characterizations. ForGH30-8 xylanases, hydrolysis of a glucuronoxylan results in singlysubstituted aldouronates of varying lengths, each containing a GAsubstitution penultimate to the reducing terminal xylan as describedabove (FIG. 1). Hydrolysis of arabinoxylan by this enzyme yields nodetectable hydrolysis products as this substrate does not contain any GAsubstitutions. Hydrolysis of these different xylans by XynA1 also yieldsresults that are expected from previous characterizations. On aglucuronoxylan, a GH10 enzyme will hydrolyze the polymer primarily toxylobiose with smaller amounts of xylose and xylotriose along with theprimary limit aldouronate product, aldotetrauronate (FIG. 21A).

Example 4 Selection of GH30-8 Xylanases

We sought to select several additional GH30-8 xylanases of the presentinvention and confirm their xylan hydrolysis product profiles. Based onqualitative assessment of the uniqueness of the amino acid sequence ofthe β7-α7 and β8-α8 loop regions as observed in FIG. 5, we selectedUniProt accession numbers C0IQA1, H1YFT8 and E4T705 for furthercharacterization. The DNA sequence of the GH30-8 with accession numberC0IQA1 derived from the nematode Meloidogyne incognita (Mi, MiXyn30A)was synthesized for cloning into an expression construct. This was doneby the company DNA2.0 (Menlo Park, Ca). The DNA coding sequence wasoptimized for expression in E. coli containing no secretion signalsequence, with addition of a C-terminal His tag for affinitypurification and the synthesized fragment cloned into their pJ411protein expression vector. For the protein coding sequence withaccession number H1YFT8 which derives from bacterium Mucilaginibacterpaludis (Mp, MpXyn30A) and accession number E4T705 from the bacteriumPaludibacter propionicigenes (Pp, PpXyn30A), genomic DNA for these twobacteria was ordered from DSMZ microbial stock center in Germany. Thegenes coding for these two enzymes were PCR amplified and the productscloned into the pET28 E. coli expression vector creating a fusion withthe vector encoded C-terminal His-tag for affinity purification of theprotein expression product.

In addition to these we also obtained the complete enzyme for which Q97was the representative catalytic domain. This protein is referred to asQ97_RCN as the additional C-terminal portion of the native amino acidsequence encodes a carbohydrate binding domain of family 13 (CBM13,Ricin-like domain, RCN). This expression construct was obtained fromDNA2.0 just as the original Q97 expression construct was obtain.However, this new expression construct did not include an affinitypurification tag. The Q97_RCN protein was considered important to showthat the enzyme still functions the same with respect to hydrolysisproduct profiles. Although, untested at this point, it is expect thatinclusion of the native CBM13 domain of Q97 may improve upon functionalfeatures such as hydrolysis reaction rate, better performance indisruption of insoluble xylans, and provide better commercial qualitiessuch as an increased temperature optimum and reaction stability.

For these four new protein expression constructs, expression was quicklyoptimized for different growth temperatures using laboratoryauto-induction procedures. It was shown that MiXyn30A did not result ina soluble protein expression product. This was confirmed by the lack ofa recoverable, near-pure protein of the correct molecular weight size bynickel immobilized metal affinity chromatography (IMAC) followed bySDS-PAGE (FIG. 19). Just to verify, the “peak region” for this IMACelution was desalted and concentrated to an estimated proteinconcentration of 0.3 mg/ml. This was used in a 50 ul overnight xylandigestion reaction intended for TLC analysis (FIG. 20). For thisprotein, no hydrolysis of xylan is observed, most likely due to notobtaining a soluble MiXyn30A protein. Most likely this protein is notsoluble as it is a eukaryotic enzyme which may, in its native nematodehost, be glycosylated, a feature which may enhance solubility and otherbiophysical features. The MpXyn30A enzyme expression was also not verygood, but IMAC chromatography resulted in a relatively pure protein inthe expected size range by SDS-PAGE (FIG. 19). Hydrolysis productanalysis by TLC showed only a very low level of hydrolysis occurred inthe overnight reaction (FIG. 20). Notably, the barely detectable largeroligosaccharides produced by this enzyme hydrolysis suggest thatMpXyn30A was not an efficiently functioning xylanase and this is likelydue to the specific sequence of the β7-α7 and β8-α8 loop regions. Ouroriginal thinking, that changes to these two regions in the GA-dependent(canonical) GH30-8 xylanases could nullify the function of these enzymeswas valid. We took a risk with a stong possibility that no activitywould be detectable and discovered a novel, broad-specificity xylanaseactivity. Just opposite of this finding, the PpXyn30A protein, althoughit also did not express well, resulted in the best yield of pure proteinfollowing the IMAC purification. This is nicely seen from the SDS-PAGEanalysis (FIG. 19). Further, an overnight reaction with this proteinyielded a hydrolysis product profile nearly identical to Q97 and C7I.This again bolsters our claim regarding these GA-independent GH30-8xylanases (FIG. 20).

The Q97_RCN protein expresses the best of all the other three, catalyticdomain only proteins. Although expression is good the vast majority ofthe expressed protein at all tested expression temperatures is ininclusion bodies. Still, purification of the soluble form yielded plentyfor biochemical studies (FIG. 19). TCL analysis of a xylan hydrolysisconfirmed that the modular enzyme functioned just as the isolatedcatalytic domain of Q97 (FIG. 20).

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration from the specification andpractice of the invention disclosed herein. All references cited hereinfor any reason, including all journal citations and U.S./foreign patentsand patent applications, are specifically and entirely incorporatedherein by reference. It is understood that the invention is not confinedto the specific reagents, formulations, reaction conditions, etc.,herein illustrated and described, but embraces such modified formsthereof as come within the scope of the following claims.

SEQUENCE LISTING

This specification includes the sequence listing that is concurrentlyfiled in computer readable form. This sequence listing is incorporatedby reference herein.

1. An isolated GA-independent GH30-8 enzyme or variant thereofexhibiting xylanase activity, the enzyme or variant comprising the aminoacid sequence (W or Y)(W or F)W(I or W or F)(not R)(not R) within theβ8-α8 loop of the enzyme or variant.
 2. The isolated GA-independentGH30-8 enzyme or variant of claim 1, comprising an amino acid sequenceat least 30% similar to an amino acid sequence selected from the groupconsisting of SEQ ID NO:1 residues 33-420 (Q97TI2), SEQ ID NO:2 residues32-421 (F1TBY8), SEQ ID NO:3 beginning at residue 45 (E4T705), SEQ IDNO:4 beginning at residue 33 (H1YFT8), SEQ ID NO:32 (C0IQA1), SEQ IDNO:33 (B3TJG3), SEQ ID NO:34 (G7M3Z8), SEQ ID NO:35 (M1N0D3), SEQ IDNO:36 (F7ZYN8), and SEQ ID NO:37 (F0KEL6).
 3. The isolatedGA-independent GH30-8 enzyme or variant of claim 2, comprising an aminoacid sequence that is at least 80% similar to the selected amino acidsequence.
 4. The isolated GA-independent GH30-8 enzyme or variant ofclaim 3, comprising an amino acid sequence that at least 95% similar tothe selected amino acid sequence.
 5. The isolated GA-independent GH30-8enzyme or variant of claim 1, comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1 residues 33-420 (Q97TI2), SEQID NO:2 residues 32-421 (F1TBY8), SEQ ID NO:3 beginning at residue 45(E4T705), SEQ ID NO:4 beginning at residue 33 (H1YFT8), SEQ ID NO:32(C0IQA1), SEQ ID NO:33 (B3TJG3), SEQ ID NO:34 (G7M3Z8), SEQ ID NO:35(M1N0D3), SEQ ID NO:36 (F7ZYN8), SEQ ID NO:37 (F0KEL6), and the aminoacid sequence of COIQA2.
 6. The isolated GA-independent GH30-8 enzyme ofclaim 1, wherein the enzyme is XynQ97.
 7. The isolated GA-independentGH30-8 enzyme of claim 1, wherein the enzyme is XynC71 (aka CpXyn30A).8. A GH30-8 enzyme composition comprising: a) a first polypeptide havingxylanase activity comprising the amino acid sequence (W or Y)(W or F)W(Ior W or F)(not R)(not R); and b) a second polypeptide having xylanaseactivity comprising the amino acid sequence (W or Y)(W or F)W(I or W orF)(not R)(not R), wherein the first and second polypeptides aredifferent, and wherein the GH30-8 enzyme composition is capable ofhydrolyzing a lignocellulosic biomass material.
 9. The GH30-8 enzymecomposition of claim 8, further comprising at least one additionalprotein having enzymatic activity.
 10. The GH30-8 enzyme composition ofclaim 8, wherein the first polypeptide, the second polypeptide, or bothcomprise an amino acid sequence that at least 30% similar to an aminoacid sequence selected from the group consisting of SEQ ID NO:1 residues33-420 (Q97TI2), SEQ ID NO:2 residues 32-421 (F1TBY8), SEQ ID NO:3beginning at residue 45 (E4T705), SEQ ID NO:4 beginning at residue 33(H1YFT8), SEQ ID NO:32 (C0IQA1), SEQ ID NO:33 (B3TJG3), SEQ ID NO:34(G7M3Z8), SEQ ID NO:35 (M1N0D3), SEQ ID NO:36 (F7ZYN8), and SEQ ID NO:37(F0KEL6).
 11. The GH30-8 enzyme composition of claim 10, wherein thefirst polypeptide, the second polypeptide, or both comprise an aminoacid sequence that is at least 80% similar to the selected amino acidsequence.
 12. The GH30-8 enzyme composition of claim 11, wherein thefirst polypeptide, the second polypeptide, or both comprise an aminoacid sequence that is at least 95% similar to the selected amino acidsequence.
 13. The GH30-8 enzyme composition of claim 12, wherein thefirst polypeptide, the second polypeptide, or both comprise an aminoacid sequence selected from the group consisting of SEQ ID NO:1 residues33-420 (Q97TI2), SEQ ID NO:2 residues 32-421 (F1TBY8), SEQ ID NO:3beginning at residue 45 (E4T705), SEQ ID NO:4 beginning at residue 33(H1YFT8), SEQ ID NO:32 (C0IQA1), SEQ ID NO:33 (B3TJG3), SEQ ID NO:34(G7M3Z8), SEQ ID NO:35 (M1N0D3), SEQ ID NO:36 (F7ZYN8), SEQ ID NO:37(F0KEL6), and the amino acid sequence of COIQA2.
 14. The GH30-8 enzymecomposition of claim 8, wherein the amount of polypeptides havingxylanase activity relative to the total amount of proteins in the enzymecomposition is about 10 wt. % to about 20 wt. %.
 15. A method ofhydrolyzing or digesting a lignocellulosic biomass material comprisinghemicelluloses, cellulose, or both, comprising contacting the GH30-8enzyme composition of claim 8 with the lignocellulosic biomass mixture.16. The method of claim 15, wherein the lignocellulosic biomass mixturecomprises an agricultural crop, a byproduct of a food/feed production, alignocellulosic waste product, a plant residue, or waste paper.
 17. Themethod of claim 15, wherein the biomass material in the lignocellulosicbiomass mixture is subjected to pretreatment, wherein the pretreatmentis an acidic pretreatment or a basic pretreatment.
 18. The method ofclaim 17, wherein the pretreatment comprises a thermal, aqueous orthermomechanical pulping.
 19. The method of claim 15, wherein the GH30-8enzyme composition is used in an amount and under conditions and for aduration sufficient to convert at least 60% to 90% of the xylan in thebiomass material into xylooligosaccharides.